珍贵橙色束丝放线菌发酵产安丝菌素P-3的分离纯化工艺优化

doi:10.16085/j.issn.1000-6613.2019-0800

摘要/Abstract

摘要：

安丝菌素是从珍贵橙色束丝放线菌的发酵液中分离出来的苯安莎类抗生素，具有显著的抗肿瘤活性。常采用乙酸乙酯对发酵液进行分离萃取，然而其发酵液成分复杂，除菌丝体外还含有许多杂蛋白质及易溶于有机溶剂的色素，这对后续提取和精制有很大的影响，影响产品质量和回收率。本文通过对AP-3发酵液进行预处理，将AP-3发酵液调整pH至3后，用10g/L硅藻土辅助抽滤；收集滤液，添加20g/L活性炭于60℃吸附2h，然后用洗脱液乙酸乙酯对活性炭进行脱附处理，2h后达到动态洗脱平衡，对洗脱液采用中性氧化铝柱层析，以乙酸乙酯和石油醚作为洗脱剂进行梯度洗脱，最终获得AP-3纯度86.15%，经重结晶后得到AP-3纯品，经过HPLC检测AP-3纯度达95%。

关键词: 安丝菌素P-3, 预处理, 活性炭, 吸附, 分离纯化

Abstract:

Ansamitocin was a benzoan antibiotic isolated from the fermentation broth of microorganisms (Actinosynnema pretiosum) and had significant antitumor activity. The fermentation broth was often separated and extracted by ethyl acetate. However, the composition of fermentation broth was complex. In addition to mycelium, the fermentation broth also contained many heteroproteins and pigments that were easily soluble in organic solvents, which had a great impact on subsequent extraction and refining, affecting product quality and recovery. In this paper, the AP-3 fermentation broth was properly pretreated. The AP-3 fermentation broth was adjusted to pH 3, and then filtered with 10g/L diatomaceous earth. The filtrate was collected, and 20g/L activated carbon was added to adsorb at 60℃for 2h. The activated carbon was desorbed with ethyl acetate as the eluent, and the dynamic elution equilibrium was reached after 2h. The eluent was subjected to neutral alumina column chromatography, and washed with ethyl acetate and petroleum ether. The elution was carried out by gradient elution, and finally the purity of AP-3 was 86.15%. After recrystallized, AP- 3 pure product was obtained, and its HPLC purity reached 95%.

Key words: ansamitocin P-3, pretreatment, activated carbon, adsorption, isolation/purification

中图分类号:

TQ465.5

吴兴可,祁荃,卞婷婷,刘苏煜,谷艺明,郭静,蔡志强. 珍贵橙色束丝放线菌发酵产安丝菌素P-3的分离纯化工艺优化[J]. 化工进展, 2020, 39(3): 1122-1128.

Xingke WU,Quan QI,Tingting BIAN,Suyu LIU,Yiming GU,Jing GUO,Zhiqiang CAI. Optimization of separation and purification process of ansamitocin P-3 from fermentation broth ofActinosynnema pretiosum ssp.auranticum[J]. Chemical Industry and Engineering Progress, 2020, 39(3): 1122-1128.

图/表 13

图1 安丝菌素结构式

图2 AP-3的HPLC标准曲线

图3 硅藻土添加量对发酵液中杂质的影响CL—抽滤； LX—离心

图4 pH对发酵液的影响

图5 脱色波长的确定

表1 不同波长下的相关系数

波长/nm	线性关系
380	R²=0.9977
400	R²=0.9879
420	R²=0.9994
440	R²=0.9988
460	R²=0.9917

图6 活性炭加入量对发酵液的影响

图7 脱色温度对发酵液的影响

图8 脱色时间对发酵液的影响

表2 不同洗脱剂对活性炭吸附安丝菌素的洗脱效果

洗脱剂	AP-3一次性洗脱率/%	色素洗脱率/%
乙酸乙酯	81.28	0
乙醇	18.64	94.16
甲醇	3.6	95.23

图9 活性炭吸附安丝菌素的洗脱过程

表3 中性氧化铝柱层析梯度洗脱消耗溶剂体积

V(乙酸乙酯)∶V(石油醚)	体积/mL
5∶1	60
3∶1	140
2∶1	180
1∶1	140
1∶2	120
0∶1	80

图10 纯化所得AP-3的MS、1H NMR图

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